DESCRIPTION (the applicant's description verbatim): ATP-sensitive (KATP) channels couple energy metabolism and stress to membrane excitability and are highly expressed in heart. Their function in the heart and coronary vasculature has been studied extensively by the use of pharmacological agents that open or block KATP channels. The molecular cloning of subunits of KATP channel revealed that KATP channels consist of pore-forming Kir6 subunits in association with regulatory SUR subunits. There are two Kir6 members (Kir6.1 and Kir6.2) - both are highly expressed in heart. Cardiac KATP channels are believed to consist of Kir6.2/SUR2 complexes. However, the role of Kir6.1 is unknown. The overall goal of this application is to examine the function of Kir6.1 subunits in the cardiovascular system. Since the localization of a protein can be indicative of its function, we will examine the regional, cellular and subcellular distribution patterns of Kir6.1 subunits in cardiac myocytes, the conduction system, in the coronary vasculature and in endothelial cells using immunohistochemistry techniques (we have developed excellent antibodies for this purpose). We show biochemically and electrophysiologically that Kir6.1 and Kir6.2 subunits can co-assemble in heterologous expression systems, suggesting that this may also occur in heart. We will examine whether such co-assembly takes place for native Kir6 proteins. We will also investigate the functional characteristics of heteromeric Kir6 channels using patch clamp techniques. We will generate mice with targeted disruption of the Kir6.1 gene. LoxP sites will be introduced in the Kir6.1 locus and these mice will be crossed with others overexpressing Cre in the heart. We will utilize these mice directly to examine the role of Kir6.1 subunits in the function of heart and vasculature using isolated, Langendorff-perfused heart techniques. Coronary blood flow and -reserve will be measured along with heart function during ischemia, reperfusion and ischemic preconditioning. These studies will provide a framework in which to understand the complexity of KATP channels in the cardiovascular system, in particular the role of Kir6.1 subunits, and will provide molecular insights into the function of KATP channels in animals during normal and pathophysiological conditions.